This invention relates to fatty acid biosynthesis and, in particular, to the preparation and use of nucleic acid fragments encoding plant fatty acid modifying enzymes associated with modification of the delta-9 position of fatty acids and, in particular, formation of conjugated double bonds. Chimeric genes incorporating such nucleic acid fragments and suitable regulatory sequences can be used to create transgenic plants having altered lipid profiles. This invention also relates to the preparation and use of nucleic acid fragments encoding plant fatty acid modifying enzymes associated with the formation of a trans-delta-12 double bond. Chimeric genes incorporating such nucleic acid fragments and suitable regulatory sequences can be used to create transgenic plants having altered lipid profiles.
Fatty acids bearing chemical modifications in addition to the common double bonds are found in the storage lipids of many oilseeds (Badami and Patil (1981) Prog. Lipid Res. 19:119-153). Some of these modifications functionalize the fatty acid to produce products that are useful in industrial applications; this is an alternative to the more common usage of plant-derived lipids as foods. Examples are the use of the hydroxylated fatty acid ricinoleic acid in lubricants, and the short- or medium-carbon chain length fatty acids from palm oil in detergents. In some cases, fatty acid composition of the storage lipids of oilseeds produced in temperate climates can be modified by the addition of genes from exotic sources so that large amounts of unique fatty acids are produced (Ohlrogge, J. B. (1994) Plant Physiol. 104, 821-826).
Fatty acids containing conjugated double bonds are major components of the seed oil of a limited number of plant species. For example, calendic acid (8-trans, 10-trans, cis-12-octadecatrienoic acid) composes greater than 50% of the total fatty acids of the seed oil of Calendula officinalis (Crombie and Holloway (1984) J. Chem. Soc. Chem. Commun. 15, 953-955, Chisholm, M. J. and Hopkins, C. Y. (1967) Can. J. Biochem 45:251-254). Another example, xcex1-parinaric acid (9-cis, 11-trans, 13-trans, 15-cis-octadecatetraenoic acid) and xcex2-parinaric acid (9-trans, 11-trans, 13-trans, 15-cis-octadecatetraenoic acid) compose more than 25% of the total fatty acids of the seed oil of Impatiens species (Bagby, M. O., Smith, C. R. and Wolff, I. A. (1966) Lipids 1, 263-267). In addition, xcex1-eleostearic acid (9-cis, 11-trans, 13-trans-octadecatrienoic acid) and xcex2-eleostearic acid (9-trans, 11-trans, 13-trans-octadecatrienoic acid) compose  greater than 55% of the total fatty acids of the seed oil of Momordica charantia (Chisolm, M. J. and Hopkins, C. Y. (1964) Can. J. Biochem. 42, 560-564; Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). Calendic acid and eleostearic acid are both 18:3 fatty acids, like linolenic acid, however, their structures are quite different, as shown in FIG. 1. Another fatty acid containing conjugated double bonds is found in the seeds of Dimorphotheca sinuata. This unusual C18 fatty acid, dimorphecolic acid (9-OH-18:2xcex9410trans,12trans), contains two conjugated trans-double bonds between the xcex9410 and xcex9411 carbon atoms and between the xcex9412 and xcex9413 carbon atoms as well as a hydroxyl group on the xcex949 carbon atom [Binder, R. G. et al., (1964) J. Am. Oil Chem. Soc. 41:108-111; Morris, L. J. et al., (1960)J. Am. Oil Chem. Soc. 37:323-327]. Thus, there are certain 18:2 and 18:3 plant fatty acids that contain conjugated double bonds.
The presence of conjugated double bonds in fatty acids provides the functional basis for drying oils such as tung oil that are enriched in isomers of eleostearic acid. This is due largely to the fact that fatty acids with conjugated double bonds display high rates of oxidation, particularly when compared to polyunsaturated fatty acids with methylene interrupted double bonds. Drying oils, such as tung oil, are used as components of paints, varnishes, and inks.
Conjugated fatty acids can also be used as an animal feed additive. Conjugated linoleic acids (CLAs, 18:2) have been used to improve fat composition in feed animals.
U.S. Pat. No. 5,581,572, issued to Cook et al. on Dec. 22, 1998, describes a method of increasing fat firmness and improving meat quality in animals using conjugated linoleic acds.
U.S. Pat. No. 5,554,646, issued to Cook et al. on Sep. 10,1996, describes a method of reducing body fat in animals using conjugated linoleic acids.
U.S. Pat. No. 5,519,451, issued to Cook et al. on Jul. 6, 1999, describes a method of improving the growth or the efficiency of feed conversion of an animal which involves animal feed particles having an inner core of nutrients and an outer layer containing a conjugated fatty acid or an antibody that can protect the animal from contacting diseases that can adversely affect the animal""s ability to grow or efficiently convert its feed into body tissue.
U.S. Pat. No. 5,428,072, issued to Cook et al. on Jun. 27, 1995, describes a method of enhancing weight gain and feed efficiency in animals, which involves the use of conjugated linoleic acid.
The mechanism by which these effects are realized is not known. It is believed that no one heretofore has discussed the use of conjugated 18:3 fatty acids (conjugated linolenic acids or ClnAs), for improving animal carcass characteristics.
The biosynthesis of fatty acids with conjugated double bonds is not well understood. Several reports have indicated that conjugated double bonds are formed by modification of an existing double bond (Crombie, L. and Holloway, S. J. (1985) J. Chem. Soc. Perkins Trans. I 1985, 2425-2434; Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). For example, the double bonds at the 11 and 13 carbon atoms in eleostearic acid have been shown to arise from the modification of the xcex9412 double bond of linoleic acid (18:2xcex949,12) (Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). The exact mechanism involved in conjugated double formation in fatty acids, however, has not yet been determined. Fatty acid desaturase (Fad)-related enzymes are responsible for producing 18:3xcex949,11,13 oils such as xcex1 and xcex2-eleostearic acid and 18:4xcex949,11,13,15 oils such as xcex1 and xcex2-parinaric acid in Impatiens, Momordica, and Chrysobalanus. Insertion of a chimeric gene comprising an isolated nucleic acid fragment encoding these enzymes into species that do not normally accumulate conjugated double-bond containing; fatty acids resulted in production of eleostearic and/or parinaric acids (Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96:12935-12940; and WO 00/11176, published on Mar. 2, 2000, the disclosure of which is hereby incorporated by reference). The present invention extends this work by answering whether 18:3xcex948,10,12 fatty acids like calendic or dimorphecolic acids can also be produced in transgenic plants. Unlike the Fad-related enzymes that modify the delta-12 position to produce eleostearic and parinaric acids, the enzymes of the present invention (with one exception as is discussed below with respect to DMFad2-1) modify the delta-9 position of fatty acids to produce calendic and dimorphecolic acids. One enzyme is disclosed herein which is associated with the formation of a trans-delta-12 double bond. The product of this enzymatic reaction then becomes the substrate for a reaction involving conjugated double bond formation comprising a delta-9 position of fatty acids. Isolation and characterization of two Calendula cDNAs, two Dimorphotheca cDNAs, and expression of a chimeric transgene, are described herein.
This invention concerns an isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation comprising a delta-9 position of fatty acids wherein said fragment or a functionally equivalent subfragment thereof (a) hybridizes to any of the nucleotide sequences set forth in SEQ ID NOs:1, 3, or 12 under conditions of moderate stringency or (b) is at least 40% identical to a polypeptide encoded by any of the nucleotide sequences set forth in SEQ ID NOs:1, 3, or 12 or a functionally equivalent subfragment thereof as determined by a comparison method designed to detect homologous sequences.
In a second aspect, this invention concerns an isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation comprising a delta-9 position of fatty acids wherein said fragment, or a functionally equivalent subfragment thereof, encodes a protein comprising any one of the amino acid sequences set forth in SEQ ID NOs:2, 4, or 13.
In a third aspect, this invention concerns a chimeric gene comprising such isolated nucleic acid fragments, or a functionally equivalent subfragment thereof, or a complement thereof, operably linked to suitable regulatory sequences.
In a fourth aspect, this invention concerns a transformed host cell or plant comprising such a chimeric gene.
In a fifth aspect, this invention concerns a method of altering the level of fatty acids in a host cell or plant wherein said fatty acids comprise a modification at a delta-9 position, said method comprising:
(a) transforming a host cell or plant with a chimeric gene as discussed above;
(b) growing the transformed host cell or plant under conditions suitable for the expression of the chimeric gene; and
(c) selecting those transformed host cells or plants having altered levels of fatty acids with double bonds.
In a sixth aspect, this invention concerns a method for producing seed oil containing fatty acids comprising a modified delta-9 position in the seeds of plants which comprises:
(a) transforming a plant cell with such a chimeric gene;
(b) growing a fertile mature plant from the transformed plant cell of step (a);
(c) screening progeny seeds from the fertile plants of step (b) for altered levels of fatty acids comprising a modified delta-9 position; and
(d) processing the progeny seed of step (c) to obtain seed oil containing altered levels of plant fatty acids comprising a modified delta-9 position.
In a seventh aspect, this invention concerns a method for producing plant fatty acid modifying enzymes associated with modification of a delta-9 position of fatty acids which comprises:
(a) transforming a microbial host cell with the claimed chimeric genes;
(b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and
(c) selecting those transformed host cells containing altered levels of protein encoded by the chimeric gene.
In an eighth aspect, this invention concerns a method to isolate nucleic acid fragments and functionally equivalent subfragments thereof encoding a plant fatty acid modifying enzyme associated with modification of a delta-9 position of fatty acids comprising:
(a) comparing SEQ ID NOs:2, 4, or 13 and other plant fatty acid modifying enzyme polypeptide sequences;
(b) identifying conserved sequences of 4 or more amino acids obtained in step (a),
(c) designing degenerate oligomers based on the conserved sequences identified in step (b); and
(d) using the degenerate oligomers of step(s) to isolate sequences encoding a plant fatty acid modifying enzyme or a portion thereof associated with modification of the delta-9 position of fatty acids by sequence dependent protocols.
In an ninth aspect, this invention concerns an isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme wherein said enzyme modifies a delta-9 position of fatty acids and further wherein said fragment or a functionally equivalent subfragment thereof (a) hybridizes to any of the nucleotide sequences set forth in SEQ ID NOs:1, 3, or 12 under, conditions of moderate stringency or (b) is at least 40% identical to a polypeptide encoded by any of the nucleotide sequences set forth in SEQ ID NOs:1, 3, or 12 or a functionally equivalent subfragment thereof as determined by a comparison method designed to detect homologous sequences.
In an tenth aspect, this invention concerns an isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme wherein said enzyme modifies a delta-9 position of fatty acids and further wherein said fragment or a functionally equivalent subfragment thereof encodes a protein comprising any one of the amino acid sequences set forth in SEQ ID NOs:2, 4, or 13.
In a eleventh aspect, this invention concerns isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme wherein said enzyme modifies a delta-9 position of fatty acids wherein said fragment or a functionally equivalent subfragment thereof (a) hybridizes to the isolated nucleic acid fragment of claim 2 under conditions of moderate stringency or (b) is at least 40% identical to a polypeptide encoded by any of the isolated nucleic acid fragments of claim 2 or a functionally equivalent subfragment thereof as determined by a comparison method designed to detect homologous sequences.
Also of interest are chimeric genes comprising such isolated nucleic acid fragments, or a functionally equivalent subfragment thereof, or a complement thereof, operably linked to suitable regulatory sequences. Transformed host cells or plants comprising such chimeric genes are of interest. Indeed, these nucleic acid fragments can be used in any of the above-identified methods such as altering the level of fatty acids in a host cell or plant, producing plant fatty acid modifying enzymes associated with modification of a delta-9 position of a fatty acid, etc.
In a twelfth aspect, this invention concerns an animal feed comprising an ingredient derived from the processing of any of the seeds obtained from plants transformed with the chimeric genes discussed herein and a method of improving the carcass quality of an animal by supplementing the diet of the animal with such animal feeds.
In a thirteenth aspect, this invention concerns an isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with the formation of a trans delta-12 double bond wherein said enzyme modifies a delta-12 position of fatty acids and further wherein said fragment or a functionally equivalent subfragment thereof (a) hybridizes to any of the nucleotide sequences set forth in SEQ ID NO:10 under conditions of moderate stringency or (b) is at least 75% identical to a polypeptide encoded by any of the nucleotide sequences set forth in SEQ ID NO:10 or a functionally equivalent subfragment thereof as determined by a comparison method designed to detect homologous sequences.
Also of interest are chimeric genes comprising such isolated nucleic acid fragments, or a functionally equivalent subfragment thereof, or a complement thereof, operably linked to suitable regulatory sequences. Transformed host cells or plants comprising such chimeric genes are of interest. Indeed, these nucleic acid fragments can be used in any of the above-identified methods such as altering the level of fatty acids in a host cell or plant, producing plant fatty acid modifying enzymes associated with modification of a delta-12 position of a fatty acid, etc.